Panax Notoginseng Saponins Improve the Erectile Dysfunction in Diabetic Rats by Protecting the Endothelial Function of the Penile Corpus Cavernosum

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ORIGINAL ARTICLE Panax notoginseng saponins improve the erectile dysfunction in diabetic rats by protecting the endothelial function of the penile corpus cavernosum

F Lin and X Gou

Diabetes mellitus (DM)-associated ED is predominantly due to neurovascular dysfunction mediated by nitric oxide (NO) suppression. Panax notoginseng saponins (PNS) are widely used for treating cardiovascular disease in China. The aim of this study was to evaluate the effects of PNS on penile erection and corpus cavernosum tissues in rats with diabetes-associated ED. Four weeks after PNS treatment, erectile function was assessed by intracavernous pressure (ICP) and mean arterial pressure (MAP) measurements. The level of NO, cyclic guanosine monophosphate (cGMP) and advanced glycation end products (AGEs) in cavernous tissue were assessed. Immunohistochemical staining and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) were performed for detecting endothelial NO synthase (eNOS) and apoptosis, respectively. The results show that ICP/MAP ratio was signiﬁcantly increased in high-dose (150 mg kg À 1 per day) PNS-treated group compared with the diabetic ED untreated group (DM group). Compared with the untreated group, the expression of eNOS and the levels of NO and cGMP were increased in the PNS-treated groups. Moreover, apoptosis was markedly decreased in the group that received 150 mg kg À 1 per day of PNS. These results suggest that PNS may be used for improving the ED in diabetic rats via the NO/cGMP pathway and restores the function of endothelium in corpus cavernosum.

International Journal of Impotence Research (2013) 25, 206–211; doi:10.1038/ijir.2013.19; published online 18 April 2013 Keywords: corpus cavernosum endothelium; diabetes mellitus; endothelial nitric oxide synthase; ED; NO

INTRODUCTION results in treating ED in nondiabetic ones, its efficacy is 7,8 ED is recognized as a serious and common chronic complication significantly lower in diabetic ED patients. of diabetes mellitus (DM).1 ED in diabetes patients usually starts Panax notoginseng is one of the most widely used traditional 10–15 years earlier than in the general population. Recent studies Chinese herbal medicines for the treatment of coronary heart on the molecular pathophysiologic mechanisms of diabetes- disease, atherosclerosis, cardiac angina and apoplexy in China. induced ED have suggested that this disease is predominantly due Panax notoginseng saponins (PNS) are the main active ingredient to neurovascular dysfunction. Normal erectile function primarily of Panax notosinseng, which is reported to be responsible for the depends on vasodilatation, which is mainly regulated by both therapeutic action of this medicine. Ginsenosides Rg1, Rb1 and Rd, nerve and endothelium-derived nitric oxide (NO).2 Under a normal and notoginsenoside R1 are considered to be the major 9 situation, the endothelial NO synthase (eNOS) produces a components of PNS. Numerous studies have demonstrated that physiological level of NO in endothelial cells. From a recent PNS has extensive effects on the cardiovascular system, including review, it is clear that NO production is markedly decreased, which increasing blood flow through the coronary arteries, promoting 10,11 is correlated with reduced eNOS expression in the penis of angiogenesis, inhibiting vessel restenosis after vascular intimal 12 diabetic rats, following chemical diabetogenesis.3 Previous studies injury, protecting on lipopolysaccharide-induced microcircu- 13 have shown that high blood glucose levels can increase latory disturbance, and maintaining the normal expansion and 14,15 accumulation of advanced glycation end products (AGEs) that contraction of vascular smooth muscle. In addition, PNS are responsible for low neuronal and endothelial-derived NO- improves the sensitivity of insulin and leptin, which contributes to 16 mediated relaxation of corpus cavernosum.4 When dysfunction its anti-hyperglycemic and anti-obese activities. Meanwhile, PNS occurs in penis endothelial cells, erectile function may be could enhance the mRNA expression of the most important 11 disrupted. Thus, diabetes has the potential to have an impact vascular endothelial growth factor. Recent studies have shown on all the components of the erectile response. that intracavernous injection of vascular endothelial growth factor Multiple approaches have been used to treat diabetes ED. These improves ED through restoration of the insulin-like growth factor includes diabetes control, psychosexual counseling, PDE5 inhibi- system and sex hormone receptor genes at the mRNA and tors and antihypertensive medications.5 The PDE5 inhibitors, such protein levels, and inhibition of apoptosis in corpora cavernosa in 17,18 as sildenafil, vardenafil and tadalafil, are efficacious oral medicine diabetic rats. Those effects suggest that the restoration of the for ED therapy. The efficacy of the PDE5 inhibitors is mainly due to endothelial function could be a valuable approach for treating ED counteraction of the degradation of cGMP, which is generated via in DM. In this study, we investigated the hypothesis that by NO/cGMP pathway.6 Although PDE5 inhibitor has shown good improving the erectile function in DM via the NO/cGMP pathway,

Department of Urology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China. Correspondence: Professor X Gou, Department of Urology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China. E-mail: [email protected] Received 27 April 2012; revised 7 February 2013; accepted 13 March 2013; published online 18 April 2013 Panax notoginseng saponins in treating diabetic ED F Lin and X Gou 207 PNS restores the function of endothelium in corpus cavernosum. Institute, Nanjing, China).20,21 Absorbance was measured at 550 nm in a The results suggest that PNS improves the ED in diabetic rats via microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) and the the NO/cGMP pathway and restores the function of endothelium NO level was detected according to the standard graph of sodium nitrite À 1 in corpus cavernosum. (in the range 1–10 mmole ml ).

cGMP and AGEs analysis MATERIALS AND METHODS The level of tissue-active AGEs was measured by ELISA using the Rat AGEs Experimental animals ELISA Kit, following the manufacturer’s instructions (BIOTANG, Walthem, Sprague–Dawley rats (male, 180–220 g of body weight) were purchased MA, USA). cGMP was performed by ELISA using the cGMP Parameter Assay from the Laboratory Animal Center of Chongqing Medical University, and Kit in accordance with the protocol specified by the manufacturer (R&D were housed in a specific pathogen-free environment and given ad libitum Systems, Minneapolis, MN, USA). access to food and water. All procedures were carried out in accordance with guidelines of the Chinese animal use, and were approved by the Immunohistochemistry ethics committee of Chongqing Medical University. The tissue samples were fixed, paraffin-embedded and then sectioned at After fasting for 16 h with free access of water, diabetes was induced by 5-m thickness. After dewaxing and rehydrating, the tissue sections were intraperitoneal injection of streptozotocin (STZ; Sigma Chemical, St Louis, washed with phosphate-buffered saline and treated with 3% hydrogen MO, USA, 60 mg kg À 1) in sterile citrate buffer. The normal rats of the peroxide (H2O2) for 15 min at 37 1C to inhibit endogenous peroxidase control group (n ¼ 6) were injected with an equivalent volume of citrate activity. Antigen retrieval was performed by placing the sections in citrate buffer. The rats were examined for blood glucose via tail vein using an buffer and heated for 30 min in a microwave oven. The sections were Ultra Vue glucose monitor (Johnson & Johnson, Shanghai, China) at 1 and 2 blocked in 4% goat serum for 30 min at 37 1C, followed by incubation at weeks after STZ injection. The rats were considered diabetic if their À 1 4 1C overnight with primary anti-eNOS antibody (BD Biosciences, San Jose, blood glucose levels were above 16.7 mmol l at the two examining time CA, USA; 1:100 dilution). The sections were then incubated with biotin- points. At 12 weeks of diabetes, the rats were placed in a transparent conjugated secondary antibody (Zhongshan Golden Bridge Biotechnology, observation cage, and were put in a tranquil laboratory without light for Beijing, China) for 30 min at 37 1C and subsequently avidin–biotin 10 min to allow them to adapt to the new environment. The rats were À 1 horseradish peroxidase complex for 30 min at 37 1C, and followed by injected with apomorphine (APO; Sigma Chemical, 100 mlkg ) subcuta- treatment with DAB chromogen for reaction visualization. The sections neously to the back of the neck. The frequency of penile erection of each were counterstained with diluted hematoxylin and mounted with neutral tested animal was observed and recorded by researchers for 30 min. An gum. In the negative control, the primary antibody was replaced by erection was counted only if the emergence of an engorged glans penis phosphate-buffered solution. For image analysis, five randomly selected and distal shaft was noted. The rats showing no erection were considered fields per section were photographed and recorded. Quantitative image to have ED and used for further experiments. analysis of the Â 400 magnification images was performed by computer- ized densitometry using the Image-Pro Plus image analysis software Treatment of rats with PNS (Media Cybernetics, Bethesda, MD, USA). The diabetes-associated ED rats were randomly divided into four groups (n ¼ 6 in each group): PNS50, PNS100 and PNS150 groups, which received TUNEL assay for apoptosis À 1 50, 100 and 150 mg kg per day of PNS, respectively, and the DM group To quantify apoptosis in the penile cavernous tissue, TUNEL (terminal that consists of diabetic ED rats receiving normal saline. PNS (Weikeqi deoxynucleotidyl transferase dUTP nick end labeling) assay was performed Biological Technology, Sichuan, China) was injected intraperitoneally daily on the paraffin sections using the TUNEL apoptosis detection kit (KeyGEN for 4 weeks. Control group was injected intraperitoneally once daily with biotech, Nanjing, China). Five areas were randomly selected for each tissue normal saline of the same volume. Body weight and glucose levels were section by visual observation at a magnification of Â 400 and then monitored at 2 and 4 weeks in all animals post treatment. photographed for further digital image analysis. The number of brown- stained apoptotic cells was counted together with the total number of cells Determination of erectile function in these selected fields, with the aid of the software-based cell counting program Image-Pro Plus. The percentage of apoptotic cells in the five fields Four weeks after PNS injection, the rats were anesthetized with an from each section was calculated. intraperitoneal injection of 3.5 ml of 10% chloral hydrate.19 Using a low abdominal midline incision, the right major pelvic ganglion was exposed, and the ipsilateral cavernosal nerve was identified. The skin overlying the Statistical analysis penis was sundered and both penile crura were exposed by removing part All results were analyzed by using the SSPS 15.0 software (SPSS, Chicago, of the overlying ischiocavernous muscle. To monitor intracavernosal IL, USA). Statistical analyses of all data were performed using a one-way pressure (ICP), the right crus was cannulated with a 25-G butterfly needle analysis of variance followed by Tukey’s test to determine significant À 1 with 250 U ml heparin–saline solution and connected to a pressure differences. Data were expressed as mean±s.d. For all experiments, transducer, and the right cavernosal nerve was stimulated with a bipolar Po0.05 was considered statistically significant. silver electrode. To record the mean arterial pressure (MAP), a PE-50 tube with 250 U ml À 1 heparinized saline fitted to a pressure transducer was inserted into the left carotid artery via a midline neck incision. Stimulus RESULTS parameters were 10 V, 20 Hz and 0.2 ms (wave amplitude). The stimulation at this frequency lasted for 50 s and the curve was repeated after an Body weights and blood glucose level interval of 5 min. ICP and MAP were converted from analog to digital The body weight and blood glucose level of the rats at the signals and transmitted to a data-acquisition program, and analyzed with beginning of PNS treatment and during the course of treatment BL-420 F Data Acquisition & Analysis System (Chengdu Tme Technology, are shown in Table 1. At 4 weeks of the experiments, body weight Chengdu, China). Maximum ICP and ICP/MAP ratio were recorded. increase in all diabetic ED groups was significantly lower than that of the control group at both the 2 and 4 week points (*Po0.05). NO production assay The PNS100 and PNS150 groups had significantly higher weight # The corpus cavernosum was collected from all rats and homogenized in gain than the DM group at week 4 ( Po0.05). Four weeks after cold phosphate-buffered solution at 2–8 1C to prepare a 10% (w/v) PNS treatment, the blood glucose levels in all the PNS-treated homogenates. Each homogenized penis sample was centrifuged at groups were significantly lower than in that of the DM group 1500 Â g for 15 min at 4 1C and the supernatants were recovered. The (#Po0.05). supernatant (50 ml) of each homogenized penis sample was used to detect NO by using the Nitric Oxide Assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The protein content per mg tissue was In vivo erectile function determined with a coomassie brilliant blue protein assay kit, according The results of ICP increase and ICP/MAP ratio monitored in the to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering experimental animals are shown in Figure 1. After stimulating the

& 2013 Macmillan Publishers Limited International Journal of Impotence Research (2013), 206 – 211 Panax notoginseng saponins in treating diabetic ED F Lin and X Gou 208 Table 1. General features of experimental animals before and after PNS treatment

Group Body weight (g) Blood glucose (mmol l À 1)

Initial 2 weeks Final Initial 2 weeks Final

Control 252±11 278±10 311±7 6.6±0.7 6.5±1 6.5±0.8 DM 201±12* 216±10* 214±9* 30±2.6* 30.3±2.5* 29.5±1.3* PNS50 202±12* 210±11* 212±5* 31±2.1* 29.3±2.6* 24.9±2.7*,# PNS100 201±11* 227±9* 239±12*,# 30.3±2.4* 26.4±2*,# 20.7±2.8*,# PNS150 212±±10* 238±7*,# 248±10*,# 28.9±1.5* 24.2±2.7*,# 19.9±2.2*,# Abbreviations: Control, control group; DM, diabetic ED untreated group; PNS50, diabetic ED group treated with 50 mg kg À 1 per day PNS; PNS100, diabetic ED group treated with 100 mg kg À 1 per day PNS; PNS150, diabetic ED group treated with 150 mg kg À 1 per day PNS. Data are expressed in mean±s.d. *Po0.05 versus control group; #Po0.05 versus DM group (n ¼ 6 in each group).

Figure 1. Effect of PNS on erectile function in diabetic ED rats. (a) The patterns of intracavernous pressure (ICP) in the experimental animals with electrostimulation of the cavernous nerve. The black lines represent electrical stimulation duration (50 s). (b) Dose response of PNS treatment in the increase of ICP and ratio of ICP to mean arterial pressure (MAP). Values are expressed as mean±s.d. *Po0.05.

International Journal of Impotence Research (2013), 206 – 211 & 2013 Macmillan Publishers Limited Panax notoginseng saponins in treating diabetic ED F Lin and X Gou 209 cavernosal nerve, significant decrease in ICP increase and ICP/MAP PNS100 and PNS150 groups as compared with that of the DM were observed in untreated DM group compared with the control group (*Po0.05, Figure 3a). The AGEs levels in all the PNS-treated group (*Po0.05, Figure 1b). Treatment with 100 and 150 mg kg À 1 groups were significantly decreased compared with that of the PNS resulted in significant improvement of erectile function DM group (*Po0.05, Figure 3b). However, there was no statistical compared with the untreated DM group, which were demon- difference among the three PNS-treated groups. strated by significant ICP increase and ICP/MAP (*Po0.05, Figure 1b). Changes of eNOS expression in corpora cavernosa eNOS was detected by immunohistochemistry on the corpora NO levels cavernosa of all experimental groups and the results were shown The results of NO concentrations in corpora cavernosa tissues are in Figure 4. eNOS was intensely positive in the corpus cavernosum shown in Figure 2. Compared with the control group, the DM tissues from the control group, moderate in the PNS150 group group had dramatically decreased NO level (*Po0.05). At the and weakly positive in the PNS50 and PNS100 groups, and in the same time, the PNS150 group had significantly increased NO level DM group (Figure 4a). The expression of eNOS was much higher in compared with the DM group (*Po0.05). The PNS100 group also the PNS150 group compared with the DM group (*Po0.05, showed an increased level of NO compared with that of the DM Figure 4b). group (*Po0.05). Cell apoptosis in corpora cavernosa Effect of PNS on AGEs and cGMP Compared with the control group, the apoptosis index had The cGMP and AGEs levels in corpora cavernosa tissues were significantly increased in the DM group (*Po0.05, Figure 4c). measured by ELISA, and the results were shown in Figure 3. Apoptosis index was increased in the corpora cavernosa from the Compared with the control group, the cGMP concentration had diabetic ED rats treated with all the three doses of PNS compared dramatically decreased in the DM group (*Po0.05). Simulta- with that of rats treated with vehicle (*Po0.05, Figure 4c). neously, The cGMP concentration was markedly increased in the

DISCUSSION A growing body of evidence suggests that endothelial dysfunction is highly associated to diabetes-associated ED.22,23 Although the precise mechanisms leading to endothelial dysfunction in the diabetic vasculature, including in the penis, are not fully understood, hyperglycemia-induced endothelial dysfunction is clearly recognized as the primary cause in the pathogenesis of the vascular complications of DM.24 Various techniques, such as visual assessment, scoring of various erectile parameters and video recording of the extent of the erectile response, are used to record penile erections in animal models in vivo.25,26 As a direct index, although visible penile erection has more important value to assess the context of the observed pressure change, the difﬁculty of accurate observation, especially for inexperienced trainee, limited the application of this method. Thus, we chose the recording of ICP and ICP/MAP ratio as the primary indices of erectile function in our research. Measuring ICP and ICP/MAP ratio provides an objective and accurate quantitative index to evaluate penile erections, and has been extensively used in penile erection 27,28 Figure 2. Effect of PNS on NO concentrations in corpora cavernosa research in rats. In addition, these two parameters are well tissues in diabetic ED rats. Values are expressed as mean±s.d. correlated with the visible penile erection that reﬂects erectile *Po0.05. function.29,30 In the current study, we found that the ICP/MAP ratio

Figure 3. Effects of PNS on cGMP and AGEs expression in corpora cavernosa tissues in diabetic ED rats. cGMP and AGEs were measured by ELISA. Values are expressed as mean±s.d. (a) Relative cGMP concentration. (b) Relative AGEs concentration. *Po0.05.

& 2013 Macmillan Publishers Limited International Journal of Impotence Research (2013), 206 – 211 Panax notoginseng saponins in treating diabetic ED F Lin and X Gou 210

Figure 4. Effects of PNS on the expression of eNOS and apoptosis in corpora cavernosa tissues. (a) Penile sections were immunostained for eNOS. Endothelial cells were stained brown. Magniﬁcation Â 400. (b) Quantitative image analysis of eNOS in corpus cavernosum. (c) TUNEL assay showing the difference of apoptosis index in the corpus cavernosum. Values are expressed as mean±s.d. *Po0.05.

and the ICP increase, as two parameters of erectile function, vascular collagen, which leads to vascular thickening, elasticity were significantly increased in the PNS150 group. Therefore, our decrease, endothelial dysfunction and atherosclerosis develop- results suggest that PNS likely restores the erectile response in the ment.37 It is suggested that AGEs contribute to diabetic ED diabetes-associated ED rats. by generating free radicals or reactive oxygen species that It has been shown that diabetes exacerbates the functional induce oxidative cell damage, NO quenching, cGMP decrease deficiency in the NO/cGMP pathway in diabetes-associated ED and cavernosal smooth muscle relaxation impairment.4 Our data patients.31 Additionally, eNOS- and nNOS-mediated cavernosal show that PNS reduced blood glucose level and corpus smooth muscle relaxation is impaired in diabetic animals.32 cavernosum AGEs. Although the blood glucose levels of experi- Evidence from both basic and clinical researches show that the mental animals did not return to the normal level in the functional deficiency of the NO/cGMP pathway markedly PNS-treated rats, a significant decrease was clearly detected. participates in the development of diabetes-associated ED. In Thus, reduction of glucose may have a role in improving diabetes- the present study, the expression of eNOS, and the levels of NO associated ED. Previous studies have indicated that PNS possesses and cGMP in corpora cavernosa tissues were markedly increased anti-hyperglycemic activity and a therapeutic potential for after administration of PNS to the diabetic ED rats. The positive hyperglycemia in type 2 diabetes through enhancing insulin- effects are likely due to the function of ginsenoside Rg1, which is stimulated glucose uptake and glycogen synthesis38 or improving one of the major active components in PNS. Rg1 was reported to insulin- and leptin sensitivity; ginsenoside Rb1 is reported to be induce eNOS phosphorylation and NO production via the non- responsible for the anti-hyperglycemic effect.16 However, in transcriptional PI3K/Akt pathway.33 Recent studies show that Rg1 this study, we used a type 1 diabetes model that is impaired in is able to downregulate miRNA-214 expression in human umbilical insulin production but not in insulin sensitivity. How PNS worked vein endothelial cells, leading to increased eNOS expression,34 in this model in suppressing glucose level is currently unknown. It enhanced NO production and the expression of eNOS mRNA is likely that other mechanisms by some PNS component(s) in tumor necrosis factor-alpha-stimulated human umbilical vein contributes to the glucose-regulating effect, which certainly needs endothelial cells.35 further investigation. Nevertheless, effectively controling AGEs and In view of the proposed mechanisms of diabetes-induced ED, glucouse may prevent progress of vascular endothelium damage. AGEs may have an important role through participating in the Previous studies have shown that the ratio of apoptotic cells in development of vascular complications in diabetes. Increased the erectile tissues in diabetic rats was significantly increased glucose level promotes the formation of AGEs, which accumulate compared with that in normal rats.39 The expression of apoptotic in the corpus cavernosal tissue, particularly in the endothelial factors, such as Bak and Bax, are increased in the diabetic rat and smooth muscle cells, and contribute to the pathogenesis penile crura.40 Recent studies show that PNS effectively protects of diabetes-associated ED.4,36 AGEs form covalent bonds with liver grafts from ischemia/reperfusion injury in rats receiving

International Journal of Impotence Research (2013), 206 – 211 & 2013 Macmillan Publishers Limited Panax notoginseng saponins in treating diabetic ED F Lin and X Gou 211 orthotopic liver transplantation, which is achieved by inhibiting 17 Shirai M, Yamanaka M, Shiina H, Igawa M, Kawakami T, Ishii N et al. Vascular the expression of tumor necrosis factor-alpha and caspase-3, and endothelial growth factor restores erectile function through modulation of the increasing the expression of Bcl-2.41 PNS has a protective effect on insulin-like growth factor system and sex hormone receptors in diabetic rat. oxidative stress-induced damage and apoptosis in cultured rabbit Biochem Biophys Res Commun 2006; 341: 755–762. bone marrow stromal cells, which is associated with scavenging 18 Yamanaka M, Shirai M, Shiina H, Tanaka Y, Enokida H, Tsujimura A et al. Vascular reactive oxygen species and regulating the Bcl-2/Bax pathway.42 endothelial growth factor restores erectile function through inhibition of apoptosis in diabetic rat penile crura. J Urol 2005; 173: 318–323. In addition, PNS promotes endothelial regeneration by regulating 19 LacKamp A, Zhang GC, Mao LM, Fibuch EE, Wang JQ. Loss of surface N-methyl-D- the expression of vascular endothelial growth factor. These aspartate receptor proteins in mouse cortical neurones during anaesthesia evidence suggest that PNS possesses an anti-apoptotic activity, induced by chloral hydrate in vivo. Br J Anaesth 2009; 102: 515–522. although the specific mechanisms in diabetic ED need further 20 Cheng F, Yu WM, Xia Y, Zhang XB, Yang SX, Ge MH. Effects of buried penis on the investigation. structure and function of corpus cavernosum in a rat model. Chin Med J (Engl) In conclusion, the present study provides evidence that PNS 2010; 123: 1736–1740. increases the expression of eNOS, controls the accumulation of 21 Feng XT, Qin CB, Leng J, Tang QL, Shi H, Zhai LN et al. Yidiyin, a Chinese herbal AGEs and inhibits cell apoptosis, suggesting that PNS could be decoction, improves erectile dysfunction in diabetic patients and rats through the used for protecting endothelial function in the penile corpus NO-cGMP pathway. Biosci Biotechnol Biochem 2012; 76: 257–263. 22 De Vriese AS, Verbeuren TJ, Van de Voorde J, Lameire NH, Vanhoutte PM. cavernosum. Treatment with PNS is likely beneficial to the Endothelial dysfunction in diabetes. Br J Pharmacol 2000; 130: 963–974. restoration of erectile response, which suggests that utilization 23 Musicki B, Burnett AL. Endothelial dysfunction in diabetic erectile dysfunction. 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